The GRAIDS Trial: a cluster randomised controlled trial of computer decision support for the management of familial cancer risk in primary care.

The objective was to evaluate the effect of an assessment strategy using the computer decision support system (the GRAIDS software), on the management of familial cancer risk in British general practice in comparison with best current practice. The design included cluster randomised controlled trial, and involved forty-five general practice teams in East Anglia, UK. Randomised to GRAIDS (Genetic Risk Assessment on the Internet with Decision Support) support (intervention n=23) or comparison (n=22). Training in the new assessment strategy and access to the GRAIDS software (GRAIDS arm) was conducted, compared with an educational session and guidelines about managing familial breast and colorectal cancer risk (comparison) were mailed. Outcomes were measured at practice, practitioner and patient levels. The primary outcome measure, at practice level, was the proportion of referrals made to the Regional Genetics Clinic for familial breast or colorectal cancer that were consistent with referral guidelines. Other measures included practitioner confidence in managing familial cancer (GRAIDS arm only) and, in patients: cancer worry, risk perception and knowledge about familial cancer. There were more referrals to the Regional Genetics Clinic from GRAIDS than comparison practices (mean 6.2 and 3.2 referrals per 10 000 registered patients per year; mean difference 3.0 referrals; 95% confidence interval (CI) 1.2-4.8; P=0.001); referrals from GRAIDS practices were more likely to be consistent with referral guidelines (odds ratio (OR)=5.2; 95% CI 1.7-15.8, P=0.006). Patients referred from GRAIDS practices had lower cancer worry scores at the point of referral (mean difference -1.44 95% CI -2.64 to -0.23, P=0.02). There were no differences in patient knowledge about familial cancer. The intervention increased GPs' confidence in managing familial cancer. Compared with education and mailed guidelines, assessment including computer decision support increased the number and quality of referrals to the Regional Genetics Clinic for familial cancer risk, improved practitioner confidence and had no adverse psychological effects in patients. Trials are registered under N0181144343 in the UK National Research Register.

Prenatal detection of unbalanced chromosomal rearrangements by array CGH.

BACKGROUND: Karyotype analysis has been the standard method for prenatal cytogenetic diagnosis since the 1970s. Although highly reliable, the major limitation remains the requirement for cell culture, resulting in a delay of as much as 14 days to obtaining test results. Fluorescent in situ hybridisation (FISH) and quantitative fluorescent PCR (QF-PCR) rapidly detect common chromosomal abnormalities but do not provide a genome wide screen for unexpected imbalances. Array comparative genomic hybridisation (CGH) has the potential to combine the speed of DNA analysis with a large capacity to scan for genomic abnormalities. We have developed a genomic microarray of approximately 600 large insert clones designed to detect aneuploidy, known microdeletion syndromes, and large unbalanced chromosomal rearrangements. METHODS: This array was tested alongside an array with an approximate resolution of 1 Mb in a blind study of 30 cultured prenatal and postnatal samples with microscopically confirmed unbalanced rearrangements. RESULTS: At 1 Mb resolution, 22/30 rearrangements were identified, whereas 29/30 aberrations were detected using the custom designed array, owing to the inclusion of specifically chosen clones to give increased resolution at genomic loci clinically implicated in known microdeletion syndromes. Both arrays failed to identify a triploid karyotype. Thirty normal control samples produced no false positive results. CONCLUSIONS: Analysis of 30 uncultured prenatal samples showed that array CGH is capable of detecting aneuploidy in DNA isolated from as little as 1 ml of uncultured amniotic fluid; 29/30 samples were correctly diagnosed, the exception being another case of triploidy. These studies demonstrate the potential for array CGH to replace conventional cytogenetics in the great majority of prenatal diagnosis cases.

Prenatal diagnosis by array-CGH.

Microscopic karyotype analysis of cultured cells has been regarded as the gold standard for prenatal diagnosis for over 30 years. Since the first application of this technique to prenatal testing in the early 1970's, this procedure has proved to be highly reliable for identifying chromosome copy number abnormalities (aneuploidy) and large structural rearrangements in foetal cells obtained invasively by either amniocentesis or chorionic villus sampling (CVS). Recognising the need for more rapid testing methods which do not require cell culture, fluorescence in situ hybridisation (FISH) and quantitative fluorescence PCR (QF-PCR) have been introduced to this field in order to answer specific diagnostic questions. However, both FISH and QF-PCR suffer the disadvantage in that they are difficult to scale to a comprehensive, genome-wide screen. Array-comparative genomic hybridisation (array-CGH) in contrast is a comprehensive, genome-wide screening strategy for detecting DNA copy number imbalances which can be rapid, less labour-intensive than karyotype banding analysis and is highly amenable to automation. Array-CGH has the potential to be used for prenatal diagnosis and may address many of the limitations of both conventional microscopic cytogenetic analyses and the more recently employed rapid-screening strategies.

Microarray based comparative genomic hybridisation (array-CGH) detects submicroscopic chromosomal deletions and duplications in patients with learning disability/mental retardation and dysmorphic features.

The underlying causes of learning disability and dysmorphic features in many patients remain unidentified despite extensive investigation. Routine karyotype analysis is not sensitive enough to detect subtle chromosome rearrangements (less than 5 Mb). The presence of subtle DNA copy number changes was investigated by array-CGH in 50 patients with learning disability and dysmorphism, employing a DNA microarray constructed from large insert clones spaced at approximately 1 Mb intervals across the genome. Twelve copy number abnormalities were identified in 12 patients (24% of the total): seven deletions (six apparently de novo and one inherited from a phenotypically normal parent) and five duplications (one de novo and four inherited from phenotypically normal parents). Altered segments ranged in size from those involving a single clone to regions as large as 14 Mb. No recurrent deletion or duplication was identified within this cohort of patients. On the basis of these results, we anticipate that array-CGH will become a routine method of genome-wide screening for imbalanced rearrangements in children with learning disability.

Predictive genetic testing in children and adults: a study of emotional impact.

AIM: To determine whether, following predictive genetic testing for familial adenomatous polyposis (FAP), children or adults receiving positive results experience clinically significant levels of anxiety or depression, and whether children receiving positive results experience higher levels of anxiety or depression than adults receiving positive results. DESIGN: Two studies, one cross sectional and one prospective. SAMPLE: 208 unaffected subjects (148 adults and 60 children) at risk for FAP who have undergone genetic testing since 1990. DEPENDENT VARIABLES: anxiety, depression; independent variables: test results, demographic measures, psychological resources (optimism, self-esteem). RESULTS: Study 1. In children receiving positive results, mean scores for anxiety and depression were within the normal range. There was a trend for children receiving positive results to be more anxious and depressed than those receiving negative results. In adults, mean scores for anxiety were within the normal range for those receiving negative results, but were in the clinical range for those receiving positive results, with 43% (95% CI 23-65) of the latter having scores in this range. Regardless of test result, adults were more likely to be clinically anxious if they were low in optimism or self-esteem. Children receiving positive or negative results did not experience greater anxiety or depression than adults. Study 2. For children receiving a positive test result, mean scores for anxiety, depression, and self-esteem were unchanged over the year following the result, while mean anxiety scores decreased and self-esteem increased after receipt of a negative test result over the same period of time. CONCLUSION: Children, as a group, did not show clinically significant distress over the first year following predictive genetic testing. Adults were more likely to be clinically anxious if they received a positive result or were low in optimism or self-esteem, with interacting effects. The association between anxiety, self-esteem, and optimism suggests that counselling should be targeted, not only at those with positive test results, but also at those low in psychological resources.

An assessment of screening strategies for fragile X syndrome in the UK.

BACKGROUND: Fragile X syndrome is an inherited form of learning disability that was defined in the late 1970s by cytogenetic detection of an associated fragile site on the X chromosome (Xq27.3). Cytogenetic estimates of the prevalence of fragile X syndrome were as high as 1 in 1039 males but have since been revised downwards. Fragile X syndrome is associated with few medical problems and the subtle physical features make clinical diagnosis difficult. The unusual pattern of inheritance, delineated in the 1980s, was explained once the fragile X syndrome gene (FMR1) had been identified in 1991. This gene contains a highly variable repeat of the nucleotide triplet, cytosine-guanine-guanine (CGG). Fragile X syndrome is caused by a large expansion of this CGG repeat (full mutation) that leads to silencing of the FMR1 gene so no gene product (FMRP) is made. This is the ultimate cause of the learning disability that, in males, is sufficient to preclude independent living. Family studies show that all individuals with a full mutation inherit it from a female (usually unaffected) who carries either a full mutation or a premutation, a smaller repeat expansion (approximately 55-200 repeats) that is unstable on female transmission. The chance of a premutation expanding to a full mutation is positively associated with the size of the repeat (approximately 95% by 90 repeats) but only for female transmissions. When a man transmits a premutation, it remains a premutation; his children are, therefore, unaffected by overt learning difficulties. The potential for population screening or systematic case-finding and extended family testing exists because every unaffected mother of an affected child has a detectable CGG repeat expansion. Reliable prenatal diagnosis is possible in males. OBJECTIVES: To assess the feasibility and acceptability of population screening by addressing the following questions in the context of existing services for families with fragile X syndrome. (1) Is there a suitable test for all fragile X genotypes? (2) What are the UK population distribution of FMR1 repeat sizes, and the prevalence of full and premutations in both sexes? (3) What reliable information, in terms of the chance of an affected child, is available to women with premutations between 55 and 200 repeats? (4) What is the effect of a premutation on the person who carries it? (5) What information is available to women with intermediate alleles of 41 to 54-60 repeats? (6) How many affected people are diagnosed? (7) Given the practice of offering extended family testing (cascade testing), what is the population prevalence of 'as-yet-undiagnosed' female carriers of a full or premutation? What proportion of women at risk can be reached by cascade testing? (8) What are the costs of fragile X syndrome to an affected person and their family and to the NHS and society? (9) What is the attitude of families to the benefits and costs of a diagnosis of fragile X syndrome, and to the prospect of population screening? (10) What data are available from existing population screening programmes? (11) What alternatives to population screening exist and are these feasible? METHODS: A key aspect of the review process was to assemble a team with extensive first-hand experience of all aspects of fragile X syndrome, including affected families and the services they use, and a wide knowledge of the relevant literature. They had followed the critical discussions at all the biennial international workshops on fragile X syndrome, including a special session at the 7th International Workshop in 1995 at which an earlier (and substantially different) draft of this report was discussed. The biomedical literature review of 2429 papers was based on MEDLINE searches, extending to PsycINFO and BIDS for the psychological aspects of [fragile X syndrome] screening. Questionnaire-based information was obtained from the UK Fragile X Society and data were collected directly from all the regional clinical genetics centres in 1995 and 1998. RESULTS: Unlike cytogenetic approaches, DNA analysis can reliably determine the FMR1 CGG repeat number and detect full mutations; however, a combination of polymerase chain reaction and Southern blotting tests is required, which limits high throughput. There are UK population-based data on FMR1 repeat sizes of up to 60 repeats but insufficient to provide a reliable estimate of the prevalence of premutations (approximately 60-200 repeats). The few data and estimates in the literature of women carriers of the premutation range from 1 in 246 to 1 in 550. Two UK DNA-based estimates of the prevalence of males with the full mutation are 1 in 4090 (Coventry) and 1 in 5530 (Wessex). There are reasonable family-based data for the risk of expansion to a full mutation for the larger premutations but in the 50-69 repeat range the estimates are less secure. (ABSTRACT TRUNCATED)

Counselling following the Prenatal Diagnosis of Klinefelter Syndrome: Comparisons between Geneticists and Obstetricians in Five European Countries.

Objective: To describe and compare the information obstetricians and geneticists in five European countries report they would give following the prenatal diagnosis of Klinefelter syndrome. Methods: 388 obstetricians and 269 geneticists from Germany, the Netherlands, Portugal, Spain and the UK completed a brief questionnaire assessing two variables: the information they reported providing to parents following the prenatal diagnosis of Klinefelter syndrome (categorized as positive or negative); and their perceptions of the quality of life with the condition. Results: Geneticists were more likely than obstetricians to report providing more positive than negative information about Klinefelter syndrome than equal amounts of positive and negative information or more negative than positive information about the condition (excess positive information). Regardless of specialty, the information that health professionals reported providing was predicted by their perceptions of the quality of life with the condition, and the country from which they came. Those perceiving quality of life as greater were more likely to provide an excess positive information, as were health professionals from Germany and the UK. Conclusions: These results suggest that the information parents across Europe receive after the prenatal diagnosis of Klinefelter syndrome varies according to the specialty and country of the health professionals consulted, and their perceptions of quality of life with the condition. This variation seems to reflect personal, cultural and professional differences between health professionals. Copyright 2002 S. Karger AG, Basel

Tyramide signal amplification (TSA) systems for the enhancement of ISH signals in cytogenetics.

This unit provides a series of techniques that dramatically improve the sensitivity of direct and indirect in situ detection. TSA is a peroxidase-based signal amplification system which is compatible with all in situ hybridization as well as immunocytochemical detection systems. The assay is performed on a glass slide and combines the use of fluorescent probes in either direct or indirect format using either an enzyme-labeled streptavidin or antibody. Protocols are provided for brightfield and fluorescent detection of single DNA targets, DNA or RNA in cultured cells, multitarget detection for DNA/RNA and DNA and RNA. In addition a protocol is included for the TSA plus system, a DNP-based detection system using horseradish peroxidase and alkaline phosphatase.

Comparative sequence analysis (CSA): a new sequence-based method for the identification and characterization of mutations in DNA.

Direct sequencing analysis is largely used to confirm and characterize mutations previously detected by more rapid tests. We have developed a method-Comparative Sequence Analysis (CSA)-that simplifies the analysis of sequencing data facilitating its use as a first screen for mutation detection. Sequence data were split into their component electrophoretograms and the use of a size standard enabled equivalent traces from different individuals to be overlaid. This allowed simple and rapid visual analysis of the results. Using this technique in a blind study, we tested 576 samples for mutations in the Von Hippel-Lindau tumor suppressor gene, VHL. We were able to identify and characterize all 78 known mutations present within the sample set (100% sensitivity and specificity).

Numbers or words? A randomized controlled trial of presenting screen negative results to pregnant women.

The Objective of this study was to test the hypothesis that presenting risk information using numbers rather than words is a more effective way of communicating the residual risk inherent in a screen negative test result. We used a randomised controlled trial in a large UK teaching hospital. Two hundred and twenty pregnant women who received negative results on serum screening for Down syndrome participated. Presentation of screen negative test results were given either as a numerical probability (e.g. you have a 1:650 chance of having a baby with Down syndrome) or as a verbal probability (your chance of having a baby with Down syndrome is low). In both interventions the verbal anchor 'it is unlikely that your baby has Down syndrome' was used. Our aims were to measure the understanding of the residual risk in a screen negative result, and anxiety. Immediately after receipt of the results, 97% of those receiving their results in the form of a numerical probability and 91% of those receiving their results in the form of a verbal probability correctly understood that their baby probably did not have Down syndrome (95% CI for difference: 0% to 12%; p=0.04). All those who were incorrect believed that their baby definitely did not have Down syndrome. Subgroup analysis showed that this effect was confined to those with lower levels of education (i.e. those without a university degree), amongst whom understanding was poorer. There was no difference between intervention groups in understanding the results at four months. There were no differences between intervention groups in the levels of anxiety at one week or four months after receiving their results. In conclusion, communicating residual risks using numbers rather than words has a small beneficial effect of increasing awareness of residual risks without raising anxiety. Further work is needed to estimate the size of this effect in less well-informed and less highly educated populations.

Detailed analysis of the oligodendrocyte myelin glycoprotein gene in four patients with neurofibromatosis 1 and primary progressive multiple sclerosis.

Neurofibromatosis 1 (NF1) is a common autosomal disorder with a wide range of neurological manifestations. The case histories of five patients, including two siblings, are reported who have both neurofibromatosis 1 and primary progressive multiple sclerosis (PPMS). A further patient with both NF1 and PPMS has since been identified. More recently, a systematic clinical review of 138 unselected adult patients with NF1 identified one patient with a slowly progressive spastic paraparesis and multiple high signal hyperintensities on T2 weighted MRI. Molecular genetic studies suggest a mechanism by which the clinical association of progressive white matter disease and NF1 might arise. The gene for NF1 is located on chromosome 17q, spans 350 kb of genomic DNA, and contains 60 exons. The gene for oligodendrocyte myelin glycoprotein (OMgp) is embedded within intron 27b of the NF1 gene. OMgp is a membrane glycoprotein that appears in the human CNS at the time of myelination. It can be detected immunohistochemically on CNS myelin and on the surface of cultured oligodendrocytes. Structurally, OMgp has the potential to function as an adhesion molecule and could contribute to the interactions between the plasma membranes of oligodendrocytes and axons required for myelination and/or axon survival. This study considers the specific hypothesis that PPMS in patients with NF1 results from concurrent mutation of the OMgp gene. The OMgp genes of four unrelated patients with NF1 and PPMS were examined using a combination of Southern blot, dosage polymerase chain reaction, and chemical cleavage of mismatch. The entire OMgp coding sequence, all intronic sequence, the intron-exon boundaries, and 1 kb of flanking sequence were screened. The DNA from two patients was found to contain an alteration in the OMgp gene resulting in an amino acid change of glycine to aspartic acid at codon 21. It is concluded that PPMS in patients with NF1 can occur without concurrent mutation of the OMgp gene. The glycine to aspartic acid polymorphic alteration at codon 21 is neither sufficient nor necessary for the development of PPMS.

Psychological consequences for parents of false negative results on prenatal screening for Down's syndrome: retrospective interview study.

OBJECTIVE: To determine the psychological consequences for parents of children with Down's syndrome of having received a false negative result on prenatal screening. DESIGN: Comparison of adjustment of parents who received a false negative result with that of parents not offered a test and those who declined a test. SETTING: Parents were interviewed in their own homes. PARTICIPANTS: Parents of 179 children with Down's syndrome (mean age 4 (range 2-6) years). MAIN OUTCOME MEASURES: Anxiety, depression, parenting stress, attitudes towards the child, and attributions of blame for the birth of the affected child. RESULTS: Overall, regardless of screening history, parents adjusted well to having a child with Down's syndrome. Compared with mothers who declined a test, mothers in the false negative group had higher parenting stress (mean score 81.2 v 71.8, P=0.016, 95% confidence interval for the difference 1.8 to 17.0) and more negative attitudes towards their children (124.9 v 134.2, P=0. 009, -16.2 to -2.4). Fathers in the false negative group had higher parenting stress test scores (77.8 v 70.0, P=0.046, 1.5 to 14.2) than fathers not offered a test. Mothers in the false negative group were more likely to blame others for the outcome than mothers who had not been offered the test (28% v 13%, P=0.032, 3% to 27%). Mothers and fathers in the false negative group were more likely to blame others for this outcome than parents who had declined a test (mothers 28% v 0%, P=0.001, 19% to 37%; fathers 27% v 0%, P=0.004, 17% to 38%). Blaming others was associated with poorer adjustment for mothers and fathers. CONCLUSIONS: A false negative result on prenatal screening seems to have a small adverse effect on parental adjustment evident two to six years after the birth of an affected child.